Asthma is one of the most common chronic diseases afflicting American children and the rate of incidence, currently 10%, has nearly doubled since 1980. Although the reason for this increase has not been determined, several genes have been implicated in the pathogenesis of this disorder. Among these is ORMDL3, a member of the ORM family of endoplasmic reticulum (ER) proteins that have recently emerged as key regulators of sphingolipid biosynthesis. The sphingolipids are a diverse family of lipids with crucialcellular functions and it is therefore not surprising that aberrant regulation of their synthesis and degradation has been associated with numerous disorders. Despite their importance, the mechanisms responsible for sphingolipid homeostasis are poorly understood. Therefore, the recent discovery that the ORM proteins exist in a complex (SPOTS) with serine palmitoyltransferase (SPT), the committed enzyme of sphingolipid biosynthesis, is very significant. Studies in yeast suggest that regulation of SPT by the ORMs is controlled by phosphorylation of their N-terminal domains and redistribution of the SPOTS complex within the ER. The redistribution is consistent with the observation that while ORM regulation of in vivo SPT activity can be easily measured, in vitro SPT activity is unaffected by the ORMs. Mammalian ORMDLs also regulate in vivo SPT activity. However, they lack the N-terminal extensions found in their yeast counterparts, suggesting a different mechanism of regulation. Using a set of highly innovative reagents and methods developed in our laboratory to study the structural organization of both yeast and human SPT, we will resolve key outstanding questions regarding the mechanism of regulation of SPT by the ORMs. First, we will determine whether SPT activity is regulated by the oligomeric state and/or redistribution of the SPOTS complex within the ER in yeast and mammalian cells. Second, we will investigate the mechanism responsible for regulation of mammalian SPT by the ORMDLs, which lack the N-terminal regulatory domain found in their yeast counterparts. The data obtained from these exploratory studies will provide important new information about the regulation of sphingolipid biosynthesis in general, and lay the groundwork for studies in animals and humans that will elucidate the role of ORMDL3 in the pathophysiology of asthma.